Morphological, molecular, and pathological characterization of didymozoid trematode infection in the nasal cavity of orange-spotted grouper (Epinephelus coioides) from Arabian Gulf waters

BACKGROUND: Didymozoid trematodes represent poorly understood parasites of marine fish worldwide, with significant knowledge gaps in the Arabian Gulf region. The taxonomic identification of didymozoids from novel anatomical sites requires integrated morphological and molecular approaches to resolve species boundaries and phylogenetic relationships. METHODS: We examined 2,530 fish specimens from nine Epinephelus species collected from Arabian Gulf waters (Saudi Arabia). Parasitological, histopathological, and ultrastructural analyses were combined with molecular characterization using 28S rDNA sequences. Phylogenetic relationships were inferred using Maximum Likelihood analysis with 1,000 bootstrap replicates. RESULTS: 52 of 500 Epinephelus coioides (10.4%) harbored distinctive golden-yellow cysts located solely in nasal cavities. No infections were found in other grouper species or anatomical sites. Histopathology revealed multiple cysts containing the didymozoid parasites, with characteristic numerous eggs; and the cysts were surrounded by thin layer of fibrous connective tissue capsule with mild host inflammatory response. Ultrastructural examination demonstrated characteristic didymozoid features including bean-shaped eggs and specialized tegument structures. Molecular analysis of four seasonal isolates yielded two haplotypes (99.81% similarity) with 1,042 nucleotide sequences deposited as GenBank accessions PQ736510-PQ736513. Phylogenetic analysis confirmed placement within Didymodiclinus (Didymozoidae), showing 99.52% sequence homology with Didymodiclininae sp. from the same host species. CONCLUSIONS: This study provides the first morphological, molecular, and histopathological characterization of nasal cavity didymozoids in E. coioides from Saudi Arabian waters of the Arabian Gulf. The infections were restricted to E. coioides among nine grouper species examined and occurred only in nasal cavities, demonstrating remarkable host and anatomical site specificity. These findings provide baseline data for didymozoid ecology in Arabian Gulf waters.